From 2012.igem.org

Realizing Flower Fairy in real world

Have you ever seen flower fairies? Probably the answer is "NO", though some of you might have come across them in your childhood, because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!! In order to realize our dream, we focused on FT protein, known as Florigen.This protein is a kind of plant hormones. First, FT proteins are produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.

Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!

When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT proteins are secreted and penetrate the cell membrane of a plant, and the plant starts blooming.

We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.
These four steps are “EXPRESSION”,”SECRETION”, ”PENETRATION”, and ”ACTIVATION”

In each step, we have some problems to be attacked.
“EXPRESSION”; It is unclear whether E.coli can express FT protein, because FT protein is derived from plant cells .
”SECRETION”; After produced, ''E.coli'' have to secrete FT protein outside of themselves.
”PENETRATION”; FT protein has to penetrate into plant cells.
”ACTIVATION”; Even if FT protein could get into the cells, it is unclear whether FT protein produced by E.coli can activate genes in shoot apex cells and induce flower formation.

1.EXPRESSION

The first step is EXPRESSION.
We had to make E.coli produce FT protein.

However, in the natural environment, wildE.coli don't have FT gene. Therefore we tried to make a new BioBrick part including FT gene and introduce it into E.coli.

Modifying FT gene for Biobrick

FT gene is derived from Arabidopsis thaliana, a model plant. Professor Araki in Kyoto University kindly gave us FT cDNA in TOPO blunt end 2(Invitrogen). FT cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified FT gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to FT. Finally, we could make new BioBrick parts of FT (Fig.1-1 C).

Fig.1-1
A : Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes.
B : Inverse PCR Method
Inverse PCR is a measure of mutating FT gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.
C : Standardization of FT cDNA. We had mutated and added prefix and suffix to FT.

Confirming expression of FT

We constructed the plasmid shown in the Fig.1-2. One was FT gene with T7 promoter ([http://partsregistry.org/Part:BBa_I719005 BBa_I719005]) and 6His tag, and the other was FT gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of E.coli, the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from E.coli using affinity chromatography.

We needed to confirm that E. coli can express FT protein, because there was a possibility that E.coli can not translate FT or FT is unstable or toxic in E.coli.
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.
As a result,　we observed FT and 6 His:FT bands　at the expected molecular weight region(Fig.1-2).
So we successfuly confirmed the FT expression!

Fig.1-2 Construction of FT generator and the results of Western blotting against FT and 6 His:FT protein
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.

We succeeded in confirming the mutation and the expression of FT and 6 His:FT protein in E.coli!

2.SECRETION

The second step is SECRETION. We want E.coli to secrete FT protein to outside of the cell.
Now our E.coli can produce FT protein, but a big issue still remains: how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables E.coli to transport FT protein continuously.

Proteins are required to go through two membranes

Fig.2-1 FT protein have two hurdles in order to get out of the cell

E.coli have two membranes: inner membrane and outer membrane. To transport FT protein to outside of E.coli, FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes.

TorA signal enables proteins to go through the inner membrane via Tat pathway

Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.

Wild E.coli have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway E.coli secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm.

We improved usability of TorA signal BioBrick

We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed.

To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly.

Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)

Fig.2-4 E.coli (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. E.coli (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.

Kil protein enables proteins to go through outer membrane

Fig.2-5 Kil protein makes holes on outer membrane.

With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of E.coli. So in our project, we introduced kil gene into E.coli. But the function of outer membrane is essential for E.coli to survive. Overexpression of kil gene, therefore, causes cell death. For this reason, we must check whether kil gene is harmful or not under our condition.

Kil protein had no significant effect on E.coli's growth

We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of E.coli. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of E.coli.

We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make E.coli secrete FT protein without cell lysis!!(Fig.2-7)

Fig.2-6 The result of evaluation of kil protein.
Vertical axis means the value of OD 600. And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.

Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.

Improvement of secretion system

Now we can let E.coli to secrete FT protein to the outside by using Tat pathway and kil protein. But E.coli secrete only a small amount of FT protein when they use Tat transporters which they inherently have. To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild E.coli have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H⁺ concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion. We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make E.coli secrete more proteins with TorA.(Fig.2-9)

Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)

Fig.2-9 pspA helps proteins to go through Tat secretion pathway.

Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)] and the sequence of pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] individually, and then, we made Tat construction composed of constitutive promoter [http://partsregistry.org/Part:BBa_J23107 (BBa_J23107)], TatABC [http://partsregistry.org/Part:BBa_K797000 (BBa_K797000)], pspA [http://partsregistry.org/Part:BBa_K797001 (BBa_K797001)] and double terminator [http://partsregistry.org/Part:BBa_B0015 (BBa_B0015)]. We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.

3.PENETRATION

The third step is PENETRATION. In order to induce flower formation, FT protein from E.coli must enter into plant cells.　This is because FT protein upregulates other proteins which lead to flower formation in plant cells.

However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells. Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.

R9 peptide enables FT protein penetrate membranes by endocytosis

Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.

R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.

This is the mechanism of how R9 peptides work(Fig3-2).

Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.
Secondly, the cell responds to this stimulus and starts to endocytose.
Finally, proteins around the endocytosing region of the cell are taken into the cell.

R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. However, there are few examples about endocytosis of plants, so we needed to check the function of R9 when used on plant cells.

Fig.3-2 Mechanism of endocytosis by R9 peptide
1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.
2.The cell responds to this stimulus and starts to endocytose.
3.FT proteins around an invaginating region of the cell are taken into the cell.

Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not.

Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins.
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.

So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency

We transformed E.coli, fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make E.coli express R9::GFP fusion protein.

In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, E.coli expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA　(Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein.

We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].

Fig.3-3 Poor growth of E.coli expressing R9::GFP. IPTG induction for the 4hours.Culture fluid of E.coli with R9::GFP fusion protein on the right hand is cloudier than that on the left.

Fig.3-4 Western blotting for checking the expression. No band of R9::GFP fusion protein. Lane1: Molecular marker Lane2: GFP([Part:BBa_I746915]) Cell lysate 10µL, not induced Lane3: GFP([Part:BBa_I746915]) Cell lysate 10µL, IPTG induced Lane4: R9::GFP Cell lysate 10µL, not induced Lane5: R9::GFP Cell lysate 10µL, IPTG induced

Fig.3-5 RT-PCR of R9::GFP Lane1:100bp ladder Lane2:GAPDH (Negative control) Lane3:GAPDH(R9::GFP) Lane4:GFP (Negative control) Lane5:GFP (R9::GFP)

We used R9 and GFP separately and to check the R9 peptides function

Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from Arabidopsis thaliana 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide

It was a question whether R9 peptides work properly and introduce FT protein into plant cells.　Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6).

First, we removed the cuticles from Arabidopsis thaliana's leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).

The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.

Fig.3-7 Verification of the R9 peptide function with use of GFP.Cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)

We can make FT protein penetrate cell membrane of plants by R9 peptide function!.

4. Activation

The final step is; Activation. We verified whether FT proteins made in E.coli work normally in plant cells.

Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT proteins.

The detail function of FT

FT proteins increase transcript activities of flowering factors which lead to flower formation in a plant cell. This proteins can .

Fig.4-1 FT proteins trigger some flowering genes and they induce flower formation. FT proteins up-regulate some flowering genes and they induce plants to bloom.

Injecting FT and verifying its function by RT-PCR

To evaluate the function of FT produced by our Fairies, we used leaf cells of Arabidopsis　thaliana , insted of cells of a shoot apex. Plants produce FT proteins in their leaves and carry them to shoot apex. Considering this system, we assumed that our Flower Fairy can make flowers bloom just by introducing active FT proteins into leaves. This way, we can get lots of samples easily as well. According to some previous researches, FT gene can also up-regulate some genes in leaves, such as FUL and SEP3. We injected FT protein into leaves and conducted RT-PCR in order to value the amount of expressed RNA.
We injected FT proteins by a syringe. We prepared two types of samples: one we treated with FT and the other treated with GFP as a control. GFP was used as a control to confirm the results of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)

Fig.4-2 injecting FT protein into plant leaves.

We performed RT-PCR to compare mRNA expression of Arabidopsis leaves treated with/without FT.

Fig.4-2 is the result of RT-PCR.

Fig.4-2 6 His tag fusion FT protein was purified with Ni-NTA agarose column.
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample. FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each. Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.) After incubation, leaves were freezed with liquid nitrogen and glinded immediately.
Total RNA was extracted by phenol-chloroform extraction. cDNA was synthesized by reverse transcription and used as templates of RT-PCR. TUBULIN was used for internal control of mRNA expression.
Lane1: TUBULIN (GFP-R9 treated) amplicon 61bp
Lane2: TUBULIN (FT-R9 treated)
Lane3: FUL (GFP-R9 treated) amplicon 132bp
Lane4: FUL (FT-R9 treated)
Lane5: SEP3 (GFP-R9 treated) amplicon 87bp
Lane6: SEP3 (FT-R9 treated)
Lane7: AP1 (GFP-R9 treated) amplicon 958bp

Fig.4-3 Electrophoresis of RNA. RNA concentration is adjusted.
Lane1: RNA of 16h incubated leaves(GFP-R9 treated)
Lane2: RNA of 16h incubated leaves(FT-R9 treated)
Lane3: RNA of fresh leaves

As shown above, we got no correct ampicon band, even from tubulin, which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.

As shown in the Fig.4-3, this time, RNA samples were degradated.
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)

Establishing an effective way of RNA purification

We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .
We improved following things;
1. Total leaf volume was increased.
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.
3. We centrifuged samples and collected supernatant twice after adding ISOGEN.
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.)
Then we used better quality of RNA for reverse transcription and retried RT-PCR. Fig.4-6 shows the result of this RT-PCR.

Fig.4-4
A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low
B: Improved RNA waveform
C:
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1

Conducting RT-PCR and qPCR again by using new RNA purification

fig

To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control. The result is shown in fig.

Checking whether FT protein really introduced or not by Western Blotting

At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. Its cause might be that FT protein was not active or decomposed in the cell, or that FT proteins did not enter plant cells in the first place. If injection process does not work well, or FT does not get into the cell, it is natural that the activity of FT does not be observed. To clarify the cause, we conducted Western Blotting on Plant leaves we had injected FT proteins.

Achievement

1.Expression

• Mutate FT sequence
• Standardize FT as an iGEM part
• Confirm expression of FT protein in E.coli

2.Secretion

• Modify TorA signal to be easy to use the signal more
• Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator
• Standardize kil gene
  • Multiply TatABC in order to strengthen Tat secretion system--not yet

3.Penetration

• Keep GFP in or around plant cells using R9 peptide
  • Introduce FT in plant cells using R9 peptide--not yet

4.Activation

• Get high quality of RNA
• Amplify genes successfully
  • Check the function of FT--not yet

At PENETRATION, GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed. It is because there is an example that protein tagged with arginine at C- terminal is correctly expressed though that at N-terminal is failed to be expressed([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ). By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve macropinocytos, a type of endocytosis.

At Activation, in order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control. It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells. If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.

If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.

When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.

When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.

If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s. Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein.

Future Works

We noticed only flowering and florigen in this time but there are many other plant hormones. We made translocation pathway from E.coli into plant cells, so we will be able to introduce plant hormones into plant cells if E.coli can make them. It means we can control plant growth in any stage through genetically engineered E.coli. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.

Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to control all living cells using this technology.

Biosafety

We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.

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